Abstract

Purpose: Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis in various cancers, including lung cancer. Hypoxia-inducible factor-1α (HIF-1α) is overexpressed in human lung cancers, particularly in non–small cell lung cancers (NSCLC), and is closely associated with an advanced tumor grade, increased angiogenesis, and resistance to chemotherapy and radiotherapy. The purpose of this study was to investigate the effects of nicotine on the expression of HIF-1α and its downstream target gene, vascular endothelial growth factor (VEGF), in human lung cancer cells.

Experimental Design: Human NSCLC cell lines A549 and H157 were treated with nicotine and examined for expression of HIF-1α and VEGF using Western blot or ELISA. Loss of HIF-1α function using specific small interfering RNA was used to determine whether HIF-1α is directly involved in nicotine-induced tumor angiogenic activities, including VEGF expression, cancer cell migration, and invasion.

Conclusion: These findings identify novel mechanisms by which nicotine promotes tumor angiogenesis and metastasis and provide further evidences that HIF-1α is a potential anticancer target in nicotine-associated lung cancer.

nicotine

nAChR

HIF-1α

VEGF

lung cancer

Worldwide, lung cancer remains the leading cause of cancer death in both men and women, with an estimated 1.2 million deaths annually, of which non–small cell lung cancer (NSCLC) accounts for 75% to 80% of deaths (1). Nicotine, a psychoactive/addictive compound in cigarette smoke and the major risk factor for lung cancer (2, 3), can promote cell proliferation in several tumor cell lines, including SCLC, NSCLC, gastric cancer, pancreatic cancer, and head and neck cancer, via multiple signaling pathways (4–8). Nicotine has been shown to protect cancer cells from apoptosis induced by diverse stimuli, such as opioid, tumor necrosis factor, UV light, chemotherapeutic drugs, and serum deprivation (5, 8–12). Moreover, several studies have reported that nicotine exerts proangiogenic activities in tumor xenografts and chick chorioallantoic membrane model of angiogenesis (6, 13–15). These studies indicate that nicotine possesses both tumor-promoting and angiogenic activities conducive to a more aggressive tumor phenotype. However, the mechanisms underlying nicotine-stimulated angiogenesis remain largely unknown.

Hypoxia-inducible factor-1 (HIF-1) activates the expression of a battery of genes involved in diverse aspects of cellular and integrative physiologic processes (16). This transcription factor consists of two subunits, HIF-1α and HIF-1β, of which HIF-1α function is tightly regulated by cellular oxygen concentration. Under hypoxic conditions, HIF-1α escapes ubiquitin degradation, forms a heterodimer with HIF-1β, binds to the cis-acting element (hypoxia-responsive elements), and activates downstream hypoxia-responsive genes (17). In addition to intratumoral hypoxia, HIF-1α activity is up-regulated by a variety of nonhypoxic signals, including the inactivation of several tumor suppressors, p53, pVHL, and PTEN (18, 19), the activation of several oncogenic pathways, Src, HER/2, and Ha-Ras (20–22), and the stimulation by certain cytokines, hormones, and growth factors (23, 24), suggesting a more ample role of HIF-1 in tumor biology.

Cumulative evidences have implied an essential role of HIF-1α pathway in tumorigenesis in several cancer types (25, 26). Clinically, high levels of HIF-1α have been detected in many solid tumors and their metastases and are closely correlated to an advanced tumor grade, an increased angiogenesis, and a resistance to chemotherapy and radiotherapy (27, 28). Similarly, increased expression of HIF-1α has been reported in NSCLC tissues with poor disease prognosis (29–31). A recent study has shown that HIF-1α activation and increased tumor metastasis were induced by a dysregulated signal transduction through the phosphatidylinositol 3-kinase (PI3K) pathway in NSCLC cells (32). These findings suggest that HIF-1α pathway is critical in tumor promotion and metastasis of NSCLC. However, the detailed molecular mechanisms underlying the overexpression of HIF-1α in NSCLC, specifically in the context of carcinogen-exposed microenvironment, such as cigarette smoke, and its contribution to human lung cancer are still poorly understood.

In this study, we have shown that nicotine, a major risk factor in lung cancer and others, significantly stimulates HIF-1α protein accumulation and VEGF expression in human NSCLC, and HIF-1α contributes, at least in part, to nicotine-enhanced in vitro tumor angiogenesis and invasion. Both proangiogenic and tumor-invasive effects induced by nicotine can be partially abrogated by treatment with small interfering RNA (siRNA) specific for HIF-1α. The mechanisms behind nicotine-induced HIF-1α expression seem to involve the nicotinic acetylcholine receptor (nAChR)-mediated activation of multiple signaling pathways.

Treatment of cells with pharmacologic inhibitors. NSCLC cells at ∼80% confluence were pretreated for 1 h with increasing concentrations of α-bungarotoxin (α-BTX), LY294002, U0126, rapamycin, GF109203X (bisindolylmaleimide I), PP2, BAPTA/AM, and W7 followed by incubation in the presence or absence of 5 μmol/L nicotine (Sigma) for 16 h. All inhibitors were purchased from Calbiochem and dissolved in DMSO (except α-BTX). The final DMSO concentration did not exceed 0.1% throughout the study. Concentrations of all inhibitors were titrated to effect without cellular toxicity as determined by trypan blue exclusion assay.

siRNA transfection assay. Chemically synthesized double-stranded siRNA specific for HIF-1α (siRNAHIF-1α), 5′-AGAGGUGGAUAUGUGUGGGdTdT-3′ and 5′-CCCACACAUAUCCACCUCUdTdT-3′, was purchased from Dharmacon Research, Inc. as described previously (33). The siRNA was transfected (200 nmol/L) using Oligofectamine reagent according to the manufacturer's instructions (Invitrogen). A nontargeting siRNA sequence (Dharmacon Research) was used as nonspecific control.

ELISA assay for VEGF production. VEGF secretion in the conditioned medium was assayed using the Human VEGF ELISA kit (PeproTech) according to the manufacturer's protocols. Results were normalized to cell counts (1 × 105).

In vitro angiogenesis assay. The in vitro angiogenesis assay kit was used according to the manufacturer's protocols (Chemicon). Human umbilical vascular endothelial cells (HUVEC; 5 × 103 per well) were seeded onto a 96-well cell culture plate coated with ECMatrix and incubated at 37°C with conditioned medium derived from siRNA-treated or nontreated A549 cells after culture in the presence or absence of 5 μmol/L nicotine for 24 h. Following 6-h incubation, capillary or tubule formation was observed under a phase-contrast microscope, and the average capillary tube branch points were enumerated in six random view fields per well.

Cell migration and invasion assay. The QCM Collagen-based Cell Invasion Assay kit was used to assess tumor cell invasion according to the manufacturer's protocol (Chemicon). Cells that migrated through the gel insert to the lower surface of the membrane were stained and photographed under a microscope. The migrated stained cells were solubilized and quantified by colorimetric measurement at 560 nm.

Statistical analysis. Data are presented as the mean ± SD for three separate experiments. One-way ANOVA and Bonferroni were used for statistical analysis using Statistical Package for the Social Sciences 11.0 for Windows software. P < 0.05 was considered to be statistically significant.

Results

Nicotine induces HIF-1α expression in human NSCLC cells. To explore the effects of nicotine on the expression of HIF-1α, A549 cells were treated with increasing concentration of nicotine for 16 h and whole-cell lysates were analyzed with Western blot. Treatment with nicotine increased HIF-1α protein expression in a concentration-dependent manner (Fig. 1A and B
). Densitometric analysis reveals a 12-fold induction of HIF-1α protein accumulation at 5 to 10 μmol/L nicotine compared with nontreatment control (P < 0.01; Fig. 1B). Parallel immunofluorescence studies showed that treatment with nicotine led to an increased accumulation of HIF-1α protein in both cytoplasm and nucleus of A549 cells, a similar effect observed on exposure to hypoxia or hypoxia mimetics, such as cobalt chloride (CoCl2; Fig. 1B). Likewise, treatment with nicotine also resulted in a concentration-dependent increase in HIF-1α protein expression in H157 cell line, a squamous cell carcinoma of lung, whereas exposure to 5 μmol/L nicotine led to 7-fold induction of HIF-1α protein accumulation (P < 0.01; Supplementary Fig. S1A).

Involvement of HIF-1α in nicotine-stimulated invasion of A549 cells. A, images photographed under a phase-contrast microscope. Magnification, ×10. A549 cells transfected with nonspecific siRNA (b and e) or specific HIF-1α siRNA (c and f) were seeded onto the QCM Collagen-based Cell Invasion Assay system and cultured the presence or absence of 5 μmol/L nicotine for 48 h, and cell migration from the upper to the lower surface of the membrane was stained and photographed using a computer imaging system, wherein nontransfected cells cultured in the presence or absence of nicotine were used as negative (a) and positive (d) controls. B, quantification of cell invasion by colorimetric measurement at 560 nm. The graph shows the relative A560 values, wherein the A560 value from negative control (a) was arbitrarily set as 1.0. **, P < 0.01 (d compared with a); *, P < 0.05 (f compared with d). Results are representative of three independent experiments. Columns, mean; bars, SD.

Discussion

Cumulative evidence indicates that nicotine, asides from its psychoactive and addictive effects, can promote tumor cell proliferation, survival, migration/invasion, and tumor angiogenesis and thus serves as a potent carcinogen (5, 6, 13, 35, 40). To date, the detailed molecular mechanisms whereby nicotine enhances tumor growth and progression remain largely unknown. A growing body of evidence implies activation of HIF-1α pathway as a critical step in carcinogenesis (41, 42) due to its linkage to several oncogenic and tumor suppressor gene pathways in cancer (43). In this study, to our knowledge, we showed for the first time that treatment with nicotine promotes HIF-1α protein accumulation (Fig. 1), which contributes, at least in part, to nicotine-induced cell invasion and in vitro tumor angiogenesis in human NSCLC cells (Figs. 5 and 6). Therefore, these findings have provided novel mechanisms of biological functions of nicotine in tumor carcinogenesis.

Nicotine exerts its biological effects by binding to the nAChRs, thus leading to the activation of a cascade of signaling pathways (44), which include the influx of Ca2+ and activation of calmodulin, PKC (40, 45), c-Src, Janus-activated kinase 2/signal transducers and activators of transcription 3 (11, 38–40), PI3K/Akt/mammalian target of rapamycin (5, 35, 46), and Raf-1/mitogen-activated protein kinase/ERK1/2 (7, 34, 45). In the present study, we also showed that nicotine activates both PI3K/Akt and ERK1/2 signaling pathways (Fig. 3) and that pharmacologically blocking the activation of nAChR-mediated signaling cascades, including the Ca2+/calmodulin, c-Src, PKC, PI3K/Akt, mitogen-activated protein kinase/ERK kinase/ERK1/2, and the mammalian target of rapamycin, significantly attenuated nicotine up-regulated HIF-1α protein accumulation and VEGF protein expression in A549 cells (Fig. 4). These results suggest that nicotine promotes HIF-1α protein accumulation and VEGF expression in human NSCLC cells by activating various downstream nAChR-mediated signaling pathways.

nAChRs were once thought to be restricted to neuronal cells, but recently, the expression of nAChR subunits has also been shown in many nonneuronal cells, including normal human bronchial epithelial cells, human lung cancer cells (5, 11, 38, 45, 46), and oral keratinocytes (39). Previous studies have reported the expression of specific α7-nAChR in several NSCLC cell lines, including A549 cells (5, 11, 38). For instance, Dasgupta et al. (11) have recently shown expression of α7-nAChR protein in A549 cells and several other NSCLC cell lines. Consistent with these findings, we also showed the expression of α7-nAChR protein in four NSCLC cell lines, including A549 cell (Supplementary Fig. S2A). In another recent study, Carlisle et al. (45) reported that functional combinations of muscle-type and neuronal nAChR subunits were expressed at both mRNA and protein levels by three lung cancer cell lines, 201T, 273T, and A549; however, none of these cell lines examined expressed α2-nAChR, α4-nAChR, and α7-nAChR proteins. The apparent controversial findings about the expression of different subunits of nAChR in lung cancer cell lines may reflect the culture conditions, growth medium, passage numbers, or inherent heterogeneity of tumor cell lines. Further studies will examine the expression profile of nAChR in several established lung cancer cell lines in parallel with human lung tumor samples.

α-BTX, a polypeptide composed of 74 amino acids containing five disulfide bridges, has been reported to block α7-nAChR–mediated downstream signaling cascades triggered by nicotine (35, 37). Reconstitution experiments in Xenopus oocytes have shown the effects of α-BTX on neuronal nAChR to be highly specific for the α7-subtype (IC50, 1.6 nmol/L) but not for the α3β4-subtype (IC50, >3 μmol/L; ref. 47). Recently, several studies have used α-BTX as a specific antagonist of α7-nAChR to block nicotine-stimulated downstream signaling pathways (35, 39, 40). However, the questions whether the muscle-type nAChR was expressed and whether α-BTX can block both α7- and muscle-type receptors were not addressed. Interestingly, Carlisle et al. (45) have recently shown that nicotine activates cell signaling pathways through both muscle-type and neuronal nAChRs in NSCLC cells, and α-BTX acts as an antagonist to both α7- and muscle-type receptors. In the present study, our results showed that pretreatment with α-BTX significantly inhibited nicotine-induced HIF-1α and VEGF expression in A549 cells (Fig. 4). However, based on recent findings by Carlisle et al. (45), more studies are needed to clarify whether α7-type, muscle-type, or both are functionally involved in nicotine-induced HIF-1α and VEGF expression in human lung cancer cells.

VEGF has been recognized as one of the principal initiators of tumor angiogenesis. VEGF expression is regulated by a plethora of external factors (48), of which hypoxia is the best-characterized mediator of VEGF secretion, whereas HIF-1 protein is stabilized and bound to the hypoxia-responsive elements on VEGF promoter, thus leading to the transcriptional activation of the VEGF gene (17, 20, 26). In addition to hypoxia-responsive elements, the promoters of VEGF share many consensus sites for Sp1/Sp3, AP-2, Egr-1, and signal transducers and activators of transcription 3 (48). Previous studies have reported that nicotine stimulates VEGF expression in cancer cells (6, 14, 49), but the underlying mechanisms remain largely unknown. In this study, we showed that nicotine significantly stimulated VEGF production, which can be partially inhibited (∼46.8%) by disrupting HIF-1α expression using siRNA strategy (Figs. 1 and 3). Meanwhile, our results indicate that use of selective pharmacologic inhibitors to block nicotine-stimulated downstream signaling pathways had a stronger overall inhibitory effect on nicotine-induced HIF-1α expression than on the expression of VEGF gene (Fig. 4). Functionally, we found that nicotine-treated lung cancer cells significantly stimulated in vitro capillary-like tubule formation by HUVECs and such stimulatory effect was partially abolished (∼37.8%) by transfection with specific HIF-1α siRNA (Fig. 5). Collectively, these findings suggest that HIF-1α contributes, at least in part, to the up-regulation of VEGF expression and the in vitro tumor angiogenesis enhanced by nicotine in lung cancer cells. Therefore, further studies are necessary to investigate whether other transcription factors, such as Sp1, AP-1, and signal transducers and activators of transcription 3, are also responsible for nicotine-stimulated VEGF expression.

There is evidence that, in addition to its critical role in angiogenesis, HIF-1α plays an equally important role in tumor metastasis (32, 42, 50). Nicotine, besides its antiapoptotic (10–12) and proangiogenic (13–15) activities, is capable to promote tumor invasion and metastasis (14, 40) mediated by matrix metalloproteinases 2 and 9 and plasminogen activators (urokinase-type plasminogen activator and its receptor; ref. 14). In addition, Xu and Deng (40) have recently reported that PKCι promotes nicotine-induced migration and invasion of human lung cancer cells via phosphorylation of μ- and m-calpains. In this study, we found that migration and invasion of A549 cells stimulated by nicotine was suppressed by ∼36.6% after disruption of HIF-1α by specific siRNA (Fig. 6), suggesting that HIF-1α contributes, at least in part, to nicotine-stimulated cell migration and invasion of lung cancer. Further studies are in progress to delineate the interaction between HIF-1α and the above-mentioned pathways involved in the biological actions of nicotine in the migration and invasion of lung cancer cells.

In summary, the study described here has shown for the first time to our knowledge that nicotine stimulates HIF-1α protein accumulation and VEGF expression in NSCLC cells. We also found that HIF-1α contributes, at least in part, to nicotine-promoted cell migration, invasion, and tumor angiogenesis by lung cancer cells. These unique findings have greatly extended our current knowledge about the molecular mechanisms underlying the tumorigenic activities of nicotine and provide potential targets for the development of novel anticancer therapy in the management of local and systemic diseases in tobacco-associated human lung cancer.

Footnotes

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